Enveloped viruses use specialized fusion proteins to enter their host cells. Merger of the viral envelope with cellular membrane leads to delivery of viral nucleocapsid into the cytosol and initiates infection. Hemagglutinin (HA) of influenza virus and gp160 of HIV are the most abundant proteins of the viral envelope. The high mutation rates of HA and gp160 complicate development of universal vaccines against these viruses. However, as proteins change their conformation during fusion, they transiently expose cryptic, highly conserved sites to neutralizing antibodies and drugs. Therefore, studies of the molecular mechanisms of the molecular mechanisms of fusion and the changes in fusion proteins are not only important from the point of view of fundamental science, but also for designing new strategies to fight viral infection. The core structures of a number of viral fusion proteins share a striking similarity: they all fold into a so-called hairpin motif. However, the relevance fo this structure to fusion is not known. The hypothesis that folding of fusion proteins into a hairpin structure leads to fusion pore formation will be tested. Cells expressing HA and gp160 will be bound and fused to target cells carrying appropriate receptors. Fluorescence microscopy and real-time electrical admittance measurements will be used to monitor the extent and the kinetics of fusion induced by these proteins. The fusion reaction will be arrested at an intermediate stage upstream of fusion pore by sub-optimal temperatures. The conformational states of HA and gp160 will be explored along with concomitant lipid rearrangements induced by these proteins. The stages at which the proteins acquired the final hairpin structure will be explored with inhibitory peptides that bind to intermediate conformations of fusion proteins and prevent hairpin formation. Amphipathic agents that selectively destabilize and convert the local merger of outer leaflets of membranes into full fusion will be used to determine whether hemifusion has occurred at the intermediate stage. This interdisciplinary approach will be used to determine the rate-limiting stages of fusion for both HA and gp160 by dissecting the temperature- dependence of early and late stages of fusion.